一个播散性卡介苗感染家系致病基因的突变分析和功能研究
发布时间：2018-11-28 10:04
【摘要】：播散性卡介苗感染(Mendelian susceptibility to mycobacterial disease,MSMD)疾病于1951年首次报道,该病的临床表型是患者对一些弱毒性分枝杆菌(例如卡介苗、非结核性的环境分枝杆菌)易感,属于一种罕见的原发性免疫缺陷疾病。本研究对一个播散性卡介苗感染家系进行了分子遗传学分析,找到了其致病基因,并通过分子生物学和生物化学等实验方法研究了该致病基因的突变是如何影响其编码蛋白质的功能,初步阐明其致病机制。家系的先证者临床诊断为播散性卡介苗感染疾病(MSMD),先症者的母亲也有类似的表型,对符合遗传规律的已知基因进行筛查,最终发现致病基因STAT1外显子15,第1228位碱基从A突变成G(c.1228AG),导致该基因编码蛋白质的第410位氨基酸由赖氨酸突变为谷氨酸(p.K410E),先症者的母亲携带同样的突变,为了进一步确定该突变为致病突变,对家系内成员以及家系外100个正常人进行了限制性片段长度多态性分析验证,证实只有患者携带该突变,正常人都不携带该突变,说明该突变不是单核苷酸多态位点。进一步通过对STAT1基因突变位点保守性及其蛋白的编码结构区域分析,发现突变的氨基酸位点在物种进化上高度保守,而且突变位点位于STAT1蛋白的DNA结合区。将野生型和突变型的STAT1基因分别构建到pRc/CMV表达载体上,转染到HEK293细胞,经干扰素IFN-γ刺激后,进行免疫荧光定位并提取细胞核内总蛋白做凝胶迁移率实验(Electrophoretic Mobility Shift Assay,EMSA)实验,结果显示突变型的STAT1蛋白入核并未受到影响,但与野生型相比,STAT1蛋白组装的gamma激活因子(gamma-activating factor,GAF)与gamma激活序列(gamma-activating sequence,GAS)结合能力显著减弱,因此STAT1基因的突变影响了其生化功能。本研究通过对一个MSMD家系进行遗传学研究发现STAT1基因的一个新的致病突变(c.1228AG,p.K410E),通过相关分子生物学和生化实验初步阐明了STAT1基因K410E突变导致MSMD疾病的生化机制,进一步加深了对STAT1基因导致MSMD疾病的认识,为该疾病的遗传诊断与治疗提供了理论支持。
[Abstract]:Disseminated BCG infection with (Mendelian susceptibility to mycobacterial disease,MSMD was first reported in 1951. The clinical phenotype of the disease is the susceptibility of patients to some weakly toxic mycobacteria, such as Bacillus Calmette-Guerin (BCG), non-tuberculous environmental mycobacterium. It is a rare primary immunodeficiency disease. In this study, molecular genetic analysis was carried out on a family with disseminated BCG infection, and its pathogenic gene was found. Molecular biology and biochemistry were used to study how the mutation of the pathogenicity gene affected the function of the encoded protein and the pathogenicity mechanism was preliminarily elucidated. The mothers of the probands who were clinically diagnosed with disseminated BCG infection (MSMD), had similar phenotypes and screened known genes according to genetic rules, and finally found the pathogenic gene STAT1 exon 15. The 1228 base mutated from A to G (c.1228AG), causing the 410th amino acid of the protein encoded by the gene to mutate from lysine to glutamic acid (p.K410E), and the mother of the first patient carried the same mutation. In order to further identify the mutation as a pathogenic mutation, the restriction fragment length polymorphism (RFLP) analysis was carried out among family members and 100 normal persons outside the family. It was confirmed that only the patient carried the mutation, and none of the normal persons carried the mutation. The mutation is not a single nucleotide polymorphic site. By analyzing the conserved mutation sites of STAT1 gene and the coding structure of its protein, it was found that the amino acid sites of the mutations were highly conserved in species evolution, and the mutation sites were located in the DNA binding region of STAT1 protein. The wild-type and mutant STAT1 genes were constructed into pRc/CMV expression vectors and transfected into HEK293 cells. After stimulation with interferon IFN- 纬, immunofluorescence localization was carried out and the total protein in the nucleus was extracted for gel mobility experiment (Electrophoretic Mobility Shift Assay,. The results of EMSA showed that the entry of mutant STAT1 protein was not affected, but the binding ability of gamma activator (gamma-activating factor,GAF) assembled by STAT1 protein to gamma activation sequence (gamma-activating sequence,GAS) was significantly decreased compared with wild type. Therefore, the mutation of STAT1 gene affects its biochemical function. In this study, a new pathogenic mutation of STAT1 gene (c. 1228 AGp.K410E) was found in a MSMD pedigree. The biochemical mechanism of MSMD disease caused by K410E mutation of STAT1 gene was preliminarily clarified by molecular biology and biochemical experiments. The understanding of MSMD disease caused by STAT1 gene was further deepened, which provided theoretical support for the genetic diagnosis and treatment of the disease.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.2;R394
本文编号：2362530
[Abstract]:Disseminated BCG infection with (Mendelian susceptibility to mycobacterial disease,MSMD was first reported in 1951. The clinical phenotype of the disease is the susceptibility of patients to some weakly toxic mycobacteria, such as Bacillus Calmette-Guerin (BCG), non-tuberculous environmental mycobacterium. It is a rare primary immunodeficiency disease. In this study, molecular genetic analysis was carried out on a family with disseminated BCG infection, and its pathogenic gene was found. Molecular biology and biochemistry were used to study how the mutation of the pathogenicity gene affected the function of the encoded protein and the pathogenicity mechanism was preliminarily elucidated. The mothers of the probands who were clinically diagnosed with disseminated BCG infection (MSMD), had similar phenotypes and screened known genes according to genetic rules, and finally found the pathogenic gene STAT1 exon 15. The 1228 base mutated from A to G (c.1228AG), causing the 410th amino acid of the protein encoded by the gene to mutate from lysine to glutamic acid (p.K410E), and the mother of the first patient carried the same mutation. In order to further identify the mutation as a pathogenic mutation, the restriction fragment length polymorphism (RFLP) analysis was carried out among family members and 100 normal persons outside the family. It was confirmed that only the patient carried the mutation, and none of the normal persons carried the mutation. The mutation is not a single nucleotide polymorphic site. By analyzing the conserved mutation sites of STAT1 gene and the coding structure of its protein, it was found that the amino acid sites of the mutations were highly conserved in species evolution, and the mutation sites were located in the DNA binding region of STAT1 protein. The wild-type and mutant STAT1 genes were constructed into pRc/CMV expression vectors and transfected into HEK293 cells. After stimulation with interferon IFN- 纬, immunofluorescence localization was carried out and the total protein in the nucleus was extracted for gel mobility experiment (Electrophoretic Mobility Shift Assay,. The results of EMSA showed that the entry of mutant STAT1 protein was not affected, but the binding ability of gamma activator (gamma-activating factor,GAF) assembled by STAT1 protein to gamma activation sequence (gamma-activating sequence,GAS) was significantly decreased compared with wild type. Therefore, the mutation of STAT1 gene affects its biochemical function. In this study, a new pathogenic mutation of STAT1 gene (c. 1228 AGp.K410E) was found in a MSMD pedigree. The biochemical mechanism of MSMD disease caused by K410E mutation of STAT1 gene was preliminarily clarified by molecular biology and biochemical experiments. The understanding of MSMD disease caused by STAT1 gene was further deepened, which provided theoretical support for the genetic diagnosis and treatment of the disease.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.2;R394
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